Removing bubbles in a microfluidic device

ABSTRACT

Methods of removing bubbles from a microfluidic device are described where the flow is not stopped. Methods are described that combine pressure and flow to remove bubbles from a microfluidic device. Bubbles can be removed even where the device is made of a polymer that is largely gas impermeable.

FIELD OF THE INVENTION

Methods of removing bubbles from a microfluidic device are describedwhere the flow is not stopped. Indeed, methods are described thatcombine pressure and flow to remove bubbles from a microfluidic device.Bubbles can be removed even where the device is made of a polymer thatis largely gas impermeable.

BACKGROUND

Bubbles inadvertently introduced into a microfluidic system cansignificantly and negatively affect device operation. It is nearlyimpossible to operate and fill these devices under bubble-freeconditions. This is especially true for microfluidic perfusion culturesystems, which typically require sterilization and pre-conditioning ofthe surface prior to cell seeding.

If the bubble makes it into the growth area, poor cell viability canresult. Bubbles are typically cytotoxic to the cells and will rupturetheir cell membranes. Moreover, bubbles can interfere with mixing andflow. As such, microfluidic systems are extremely sensitive to even asmall bubble introduced into the device at any time during cell culture.

One solution to mitigate bubble-based problems is to integratemicrofluidic features to prevent bubbles from entering critical areas ofa device. There are, in general, two different approaches: trappingversus debubbling. A bubble trap is a structure integrated into the flowsystem that halts further progress of a bubble through a device. Thetrapping approach has the advantage that device operation is maintainedwhile the bubbles are trapped. However, because the bubble trap does notremove bubbles from the system, the bubble trap can completely fill withbubbles. At this point, any additional bubbles are sent through thesystem and lead to problems. In addition, the trap may not catch all thebubbles in the system.

The alternative to the trap is the debubbling demonstrated by Kang etal. Lab Chip 8:176-178 (2008). They actively removed bubbles from thesystem. This method relies upon the gas permeability of PDMS and usespositive pressure to force bubbles out of the channel and up into thepolymer. The advantage here is that the bubbles are removed from thesystem. However, in order to achieve this, the device has to be sealed,the flow stopped, and the device pressurized to force bubbles outthrough the polymer. For a microfluidic perfusion system, this meansthat the media supply to the cells is stopped, altering the environmentcells and possibly leading to nutritional deficiencies.

What is needed is a method of removing bubbles from a microfluidicdevice where the flow is not stopped.

SUMMARY OF THE INVENTION

Methods of removing gas or air bubbles from a microfluidic device aredescribed, including one or more bubbles in a microchannel of amicrofluidic device, where the flow is not stopped. Indeed, embodimentsof methods are described that combine pressure and flow to removebubbles from a microfluidic device. Bubbles can be removed even wherethe device is made of a polymer that is largely gas impermeable, sinceembodiments of the method do not involve forcing bubbles out through thepolymer. In one embodiment, at least a portion of a microchannel istreated to make it hydrophilic (or at least more hydrophilic).

In one embodiment, the present invention contemplates a method ofreducing bubble volume, comprising: a) providing a microfluidic devicecomprising a microchannel, said microchannel comprising a bubble, saidbubble having a volume; and b) flowing fluid under pressure through saidmicrochannel under conditions such that said bubble volume is reduced.While gas permeable polymers, in a preferred embodiment saidmicrochannel is made of a polymer that is substantially gas impermeable.It is not intended that the present invention be limited to anyparticular measurement of gas impermeability; however in one embodiment,it is measured by the rate of oxygen transmission (e.g. oxygentransmission rate properties on the order of less than 0.2 cc/100in²/day, more preferably less than 0.1 cc/100 in²/day, and still morepreferably less than 0.01 cc/100 in²/day).

It is not intended that the present invention be limited to anyparticular polymer that is substantially gas impermeable. In oneembodiment, said polymer is a cyclic olefin polymer.

In one embodiment, said microchannel is in fluidic communication with afirst reservoir at a first end of said microchannel, and a secondreservoir at a second end of said microchannel.

In one embodiment, said first reservoir comprises fluid under a firstpressure and said second reservoir comprises fluid under a secondpressure, wherein said first pressure is greater than said secondpressure. In one embodiment, said microchannel is in a perfusionmanifold (and the reservoirs are in the perfusion manifold). In oneembodiment, said perfusion manifold is engaged with and in fluidiccommunication with a microfluidic chip. In one embodiment, saidperfusion manifold comprises a skirt, said skirt comprising a side trackengaging said microfluidic chip. In one embodiment, said microfluidicchip comprises one or more ports and said perfusion manifold is influidic communication with said microfluidic chip through said one ormore ports. In one embodiment, said perfusion manifold delivers fluid tosaid microfluidic chip at a flow rate through said one or more ports. Inone embodiment, said first pressure is 21 kPa and said second pressureis 20 kPa. In one embodiment, said bubble is a gas bubble. In oneembodiment, said gas is oxygen, nitrogen or a mixture thereof. In oneembodiment, said bubble is an air bubble. In one embodiment, said flowrate is 40 uL/hr. In one embodiment, said flow rate is greater than 40uL/hr. In one embodiment, said flow rate is 50 uL/hr. In one embodiment,said flow rate is between 50 and 75 uL/hr. In one embodiment, saidmicrofluidic device comprises viable cells in said microchannel and saidfluid comprises media supplied to said viable cells (e.g. via aperfusion manifold of the type shown in FIGS. 1A and 1B). In oneembodiment, said media prior to step b) was degassed. In one embodiment,said media of step b) is unsaturated. In one embodiment, said mediaprior to step b) was not degassed. In one embodiment, step b) isperformed for at least one 1 hour. In one embodiment, step b) isperformed for 2 hours. In one embodiment, the method further comprisesc) introducing fluid into said microchannel, wherein said fluid has notbeen degassed.

In yet another embodiment, the present invention contemplates a methodof reducing bubble volume, comprising: a) providing a microfluidicdevice comprising a microchannel, said microchannel made of a polymerthat is substantially gas impermeable, said microchannel comprising abubble, said bubble having a volume; and b) flowing fluid under pressurethrough said microchannel under conditions such that said bubble volumeis reduced. In one embodiment, step b) is performed for between 1 and 2hours.

In one embodiment, said microchannel is in fluidic communication with afirst reservoir at a first end of said microchannel, and a secondreservoir at a second end of said microchannel. In one embodiment, saidfirst reservoir comprises fluid under a first pressure and said secondreservoir comprises fluid under a second pressure, wherein said firstpressure is greater than said second pressure. In one embodiment, saidmicrochannel is in a perfusion manifold (e.g. containing thereservoirs). In one embodiment, said perfusion manifold is engaged withand in fluidic communication with a microfluidic chip. In oneembodiment, said perfusion manifold comprises a skirt, said skirtcomprising a side track engaging said microfluidic chip. In oneembodiment, said microfluidic chip comprises one or more ports and saidperfusion manifold is in fluidic communication with said microfluidicchip through said one or more ports. In one embodiment, said firstpressure is 21 kPa and said second pressure is 20 kPa. In oneembodiment, said bubble is a gas bubble. In one embodiment, said gas isoxygen, nitrogen or a mixture thereof. In one embodiment, said bubble isan air bubble. In one embodiment, said flowing of fluid is at a flowrate of 40 uL/hr. In one embodiment, said flow rate is greater than 40uL/hr. In one embodiment, said flow rate is 50 uL/hr. In one embodiment,said flow rate is between 50 and 75 uL/hr. In one embodiment, saidmicrofluidic device comprises viable cells in said microchannel and saidfluid comprises media supplied to said viable cells. In one embodiment,said media prior to step b) was degassed. In one embodiment, said mediaof step b) is unsaturated. In one embodiment, said media prior to stepb) was not degassed. In one embodiment, step b is performed for lessthan one hour. In one embodiment, step b) is performed for at least onehour. In one embodiment, step b) is performed for 2 hours. In oneembodiment, the method further comprises c) introducing fluid into saidmicrochannel, wherein said fluid has not been degassed.

In yet another embodiment, the present invention contemplates a methodof reducing bubble volume, comprising: a) providing a microfluidicdevice comprising a microchannel, said microchannel comprises livingcells attached thereto; b) flowing fluid at a flow rate through saidmicrochannel over said cells; c) detecting a bubble, said bubble havinga volume; and d) reducing said bubble volume with pressure withoutstopping said flowing of said fluid.

In one embodiment, said microchannel is in fluidic communication with afirst reservoir at a first end of said microchannel, and a secondreservoir at a second end of said microchannel. In one embodiment, saidbubble of step c) is positioned against a polymer that is substantiallygas impermeable. In one embodiment, said first reservoir comprises fluidunder a first pressure and said second reservoir comprises fluid under asecond pressure, wherein said first pressure is greater than said secondpressure. In one embodiment, said first pressure is 21 kPa and saidsecond pressure is 20 kPa. In one embodiment, said bubble is a gasbubble. In one embodiment, said gas is oxygen, nitrogen or a mixturethereof. In one embodiment, said bubble is an air bubble. In oneembodiment, said flow rate is 40 uL/hr. In one embodiment, said flowrate is greater than 40 uL/hr. In one embodiment, said flow rate is 50uL/hr. In one embodiment, said flow rate is between 50 and 75 uL/hr. Inone embodiment, said fluid comprises culture media supplied to saidliving cells and said cells are still living after step d). In oneembodiment, said media prior to step d) was degassed. In one embodiment,said media of step d) is unsaturated. In one embodiment, said mediaprior to step d) was not degassed. In one embodiment, step d) isperformed for at least one 1 hour. In one embodiment, step d) isperformed for 2 hours. In one embodiment, the method further comprisese) introducing fluid into said microchannel, wherein said fluid has notbeen degassed.

In yet another embodiment, the present invention contemplates a methodfor establishing a fluidic connection, comprising: a) providing a firstsubstrate comprising a first fluidic port, a second substrate comprisinga second fluidic port; b) aligning the first and second sets of fluidicports; c) contacting the first and second fluidic ports to establish afluidic connection under conditions such that a bubble forms, saidbubble having a volume; and d) flowing fluid under pressure through saidfirst or second port under conditions such that said bubble volume isreduced. In one embodiment, said first substrate comprises a guidemechanism adapted to guide the second substrate. In one embodiment, themethod further comprises prior to step b) engaging the second substratewith the guide mechanism. In one embodiment, said aligning of step b) isperformed with the guide mechanism. In one embodiment, said guidemechanism comprises a guide track positioned on said first substrate,said guide track configured to engage a portion of said secondsubstrate. In one embodiment, said bubble of step c) is positionedagainst a polymer that is substantially gas impermeable. In oneembodiment, said bubble is a gas bubble. In one embodiment, said gas isoxygen, nitrogen or a mixture thereof. In one embodiment, said bubble isan air bubble. In one embodiment, flowing of fluid is at a flow rate of30-40 uL/hr. In one embodiment, said flow rate is greater than 40 uL/hr.In one embodiment, said flow rate is 50 uL/hr. In one embodiment, saidflow rate is between 50 and 75 uL/hr. In one embodiment, said firstsubstrate comprises a channel in fluidic communication with said port.In one embodiment, said channel is a microchannel. In one embodiment,said first substrate is a perfusion manifold (e.g. of the type shown inFIGS. 1A and 1B). In one embodiment, said second substrate is amicrofluidic device. In one embodiment, said perfusion manifold engagessaid microfluidic device at step c) (e.g. as illustrated in FIGS. 1A,1B, 2C-D, or FIGS. 2E-1, 2E-2, 2E-3). In one embodiment, saidmicrofluidic device comprises a microchannel, said microchannelcomprising living cells, and said fluid comprises media supplied to saidcells. In one embodiment, said media prior to step d) was degassed. Inone embodiment, said media of step d) is unsaturated. In one embodiment,said media prior to step d) was not degassed. In one embodiment, step d)is performed for at least one 1 hour. In one embodiment, step d) isperformed for 2 hours. In one embodiment, the method further comprisese) introducing fluid into said microchannel, wherein said fluid has notbeen degassed.

In yet another embodiment, the present invention contemplates a methodof reducing bubble volume, comprising: a) providing a microfluidicdevice comprising a microchannel, said microchannel comprises livingcells attached thereto; b) flowing fluid at a flow rate through saidmicrochannel over said cells, wherein said fluid was treated prior tosaid flowing so as to render the fluid unsaturated; c) detecting abubble, said bubble having a volume; and d) reducing said bubble volumewith pressure over a period of time without stopping said flowing ofsaid fluid, wherein living cells are in said microchannel after saidperiod of time. In one embodiment, said microchannel is in fluidiccommunication with a first reservoir at a first end of saidmicrochannel, and a second reservoir at a second end of saidmicrochannel. In one embodiment, said bubble of step c) is positionedagainst a polymer that is substantially gas impermeable. In oneembodiment, said first reservoir comprises fluid under a first pressureand said second reservoir comprises fluid under a second pressure,wherein said first pressure is greater than said second pressure. In oneembodiment, the first pressure is greater by at least 0.5 kPa. In oneembodiment, said first pressure is 21 kPa and said second pressure is 20kPa. In one embodiment, said first pressure is 31 kPa and said secondpressure is 30 kPa. In one embodiment, said first pressure is 33 kPa andsaid second pressure is 32 kPa. In one embodiment, said bubble is a gasbubble. In one embodiment, said gas is oxygen, nitrogen or a mixturethereof. In one embodiment, said bubble is an air bubble. In oneembodiment, said flowing of fluid is at a flow rate of 30-40 uL/hr. Inone embodiment, said flow rate is greater than 40 uL/hr. In oneembodiment, said flow rate is 50 uL/hr. In one embodiment, said flowrate is between 50 and 75 uL/hr.

In yet another embodiment, the present invention contemplates a methodof using non-equilibrated culture media, comprising: a) providing i)non-equilibrated culture media, and ii) a microfluidic device comprisinga microchannel, said microchannel comprises living cells attachedthereto; and b) flowing said non-equilibrated culture media at a flowrate under pressure over a period of time through said microchannel oversaid cells, without stopping said flowing of said fluid, wherein livingcells are in said microchannel after said period of time and no bubblesare visible in said microchannel. In one embodiment, said microchannelis in fluidic communication with a first reservoir at a first end ofsaid microchannel, and a second reservoir at a second end of saidmicrochannel. In one embodiment, said first reservoir comprises fluidunder a first pressure and said second reservoir comprises fluid under asecond pressure, wherein said first pressure is greater than said secondpressure. In one embodiment, the first pressure is greater by at least0.5 kPa. In one embodiment, said first pressure is greater by less than2 kPa. In one embodiment, said first pressure is 21 kPa and said secondpressure is 20 kPa. In one embodiment, said first pressure is 31 kPa andsaid second pressure is 30 kPa. In one embodiment, said first pressureis 33 kPa and said second pressure is 32 kPa. In one embodiment, saidfirst pressure is 34 kPa and said second pressure is 33 kPa. In oneembodiment, said bubble is a gas bubble. In one embodiment, said gas isoxygen, nitrogen or a mixture thereof. In one embodiment, said bubble isan air bubble. In one embodiment, said flowing of non-equilibratedculture media is at a flow rate of 30-40 uL/hr. In one embodiment, saidflow rate is greater than 40 uL/hr. In one embodiment, said flow rate is50 uL/hr. In one embodiment, said flow rate is between 50 and 75 uL/hr.

In still another embodiment, the present invention contemplates, amethod of reducing bubble volume in a microfluidic device with twomicrochannels, comprising: a) providing a microfluidic device comprisingfirst and second microchannels separated by a deformable membrane,wherein a bubble is in said first or second microchannel or both, saidbubble having a volume; and b) flowing fluid under pressure through saidfirst and second microchannels under conditions such that said bubblevolume is reduced and said deformable membrane is not deformed (ordeformed less than 20%, more preferably less than 10% and mostpreferably less than 5%). In one embodiment, i) said first microchannelis in fluidic communication with a first reservoir at a first end ofsaid first microchannel, and a second reservoir at a second end of saidfirst microchannel and ii) said second microchannel is in fluidiccommunication with a third reservoir at a first end of said secondmicrochannel, and a fourth reservoir at a second end of said secondmicrochannel. In one embodiment, i) said first reservoir comprises fluidunder a first pressure and said second reservoir comprises fluid under asecond pressure, wherein said first pressure is greater than said secondpressure and ii) said third reservoir comprises fluid under a firstpressure and said fourth reservoir comprises fluid under a secondpressure, wherein said first pressure is greater than said secondpressure. In one embodiment, said first pressure is 21 kPa and saidsecond pressure is 20 kPa. In one embodiment, said first pressure is 31kPa and said second pressure is 30 kPa. In one embodiment, said firstpressure is 33 kPa and said second pressure is 32 kPa. In oneembodiment, said first pressure is 34 kPa and said second pressure is 33kPa. In one embodiment, said second reservoir and said fourth reservoirshare a pressure regulator (in order to maintain equal, or very nearlyequal, pressures within the two microchannels).

In preferred embodiments, the present invention contemplates utilizingnon-equilibrated and non-degassed culture media with microfluidicdevices. In one embodiment, the present invention contemplatesequilibrating via the process of degassing (physically removingdissolved gas from solution) media before a first pressure/flowcycle—but using non-equilibrated and non-degassed media when replacingmedia thereafter, i.e. during long-term culture. That is to say, culturemedia is equilibrated and/or de-gassed once, e.g. at the beginning ofthe experiment, and then a pressure/flow treatment is utilized for aperiod of time. In another preferred embodiment, the present inventioncontemplates using non-equilibrated and non-degassed media even in afirst pressure/flow cycle (albeit with higher pressures) wheneverculture media is placed into the perfusion manifold or “pod”reservoir(s). In this embodiment, culture media is not equilibrated(i.e. it is non-equilibrated culture media) and has not gone thephysical removal of dissolved gas via degassing.

It has been found empirically that 1) cells (including cells sensitiveto shear forces such as motor neurons) are capable of handling elevatedflow rates, i.e. flow rates that help to facilitate bubble removal,without loss of viability or inhibition of development (e.g. noinhibition of axon growth), 2) capable of handling multiplepressure/flow cycles at 20 kPa applied pressure and that 3) the use ofcold media to refill inlet reservoirs during normal mediarefresh/addition steps did not cause the formation of bubbles after theinitial pressure/flow step to remove system bubbles.

DESCRIPTION OF THE INVENTION

In one embodiment, the present invention contemplates putting amicrofluidic device in fluidic communication with another microfluidicdevice, including but not limited to, putting a microfluidic device influidic communication with the perfusion manifold assembly.Unfortunately, putting devices in fluidic communication with each othercan result in the formation of bubbles (40), as shown schematically inFIGS. 3A and 3B. These can also be trapped when initially filling agas/air filled chip with fluid. Air bubbles are particularly challengingin microfluidic geometries because they get pinned to surfaces and arehard to flush away with just fluid flow. They pose additional challengesin cell culture devices because they can damage cells through variousmeans.

Moreover, bubbles may grow. For example, they may grow because ofequilibration with 5% CO2 and a humid environment. They may grow becauseof capillary force from hydrophobic surfaces. On the other hand, theymay grow because of an oversaturated media due to a pressure drop withinthe perfusion disposable (“PD”).

As noted above, one approach to removing bubbles is the debubblingdemonstrated by Kang et al. Lab Chip 8:176-178 (2008). They activelyremoved bubbles from the system by utilizing the gas permeability ofPDMS; positive pressure was used to force bubbles out of the channel andup into the polymer. The advantage here is that the bubbles are removedfrom the system. However, in order to achieve this, the device has to besealed, the flow stopped, and the device pressurized to force bubblesout. For a microfluidic perfusion system, this means that the mediasupply to the cells is stopped, altering the environment cells andpossibly leading to nutritional deficiencies.

In addition, the Kang et al. approach relies on the gas permeabilityPDMS. While PDMS is commonly used in microfluidics, there are goodreasons for not using such gas permeable materials, i.e. good reasonsfor using materials that are substantially not gas permeable in a chip.First, it can be difficult to control the gas content of liquids presentin a chip if the surrounding material is gas permeable, as the liquidmay gain or lose gas content through the gas permeable material. Thiscan be relevant, for example, where one wants to model hypoxicconditions, e.g. hypoxic conditions present in some portions of theintestinal tract (modeled by the so-called “gut-on-chip.”) Second, gaspermeability can exacerbate bubbles, as bubble can gain gas through thegas permeable material. Third, gas permeable materials often alsopossess higher gas-carrying capacity, which can fuel bubbles even in theabsence of convective gas transport. Fourth, materials that arepermeable to gasses such as oxygen are often also more permeable towater vapor. Accordingly, gas permeability of surrounding material canlead to evaporation from the microfluidic device.

While there are good reasons for not using materials such as PDMS, thereis more to consider. Materials that may happen to be substantially gasimpermeable can be favored for other reasons. For example, COP (cyclicpolyolefin), polycarbonate, acrylic or polystyrene materials may beselected due to their compatibility with injection molding, opticalclarity, strength or a variety of other parameters. These materials tendto be substantially gas impermeable (at least at typical thicknesses andin comparison with the gas permeability of PDMS), but their selection isbased on other factors.

In any event, the use of materials that may happen to be substantiallygas impermeable makes the debubbling approach of Kang et al. unworkable.The bubbles will not be driven into the polymer.

Of course, one approach is to make the conditions less likely forgenerating bubbles. For example, one approach is to make the fluid layerhydrophilic or more hydrophilic. This reduces the chance of trappedbubbles during priming. Moreover, bubbles should want to shrink normallyif media is at equilibrium.

But once there are bubbles, the present invention contemplates activereduction and/or removal using a combination of pressure and flow. Inone embodiment, two reservoirs are employed. One can then utilize eithera push based flow method (FIG. 6) or a pull based flow method (FIG. 7).In the pull based flow method, oversaturated media won't be in thecritical areas of system. This requires swapping positive pressureregulators with vacuum regulators.

A preferred method, however, utilizes a pressure differential and flow.As shown in FIG. 8, even small pressure differentials (P₁ versus P₂)result in good pressure (sufficient to reduce bubbles) without requiringunrealistic flow rates. In this method, going below a certain appliedpressure results in very long (impractical) periods of time to reducethe bubble volume. Moreover, utilizing such low pressure makes thesystem sensitive to small changes and inconsistencies.

This does not mean that very high pressures need to be used. Indeed,above a certain pressure there are only diminishing returns, i.e. ittakes about the same amount of time (short period) to reduce the bubblevolume.

While it is not intended that the present invention be limited to anyparticular mechanism, it is believed that a) the bubble shrinks due toequilibration with dissolved gas in the media, b) there is insignificantcapillary pressure to cause the bubble to shrink, c) there isinsignificant vapor pressure so as to cause the bubble to grow, and d)there is no gas permeation through either the chip or the perfusiondisposable. Said another way, where the media passing by the bubble isunsaturated or under-saturated, it has the ability to take in/dissolvegas from the bubble. One can increase the amount or volume of gas thatthe media can consume (dissolve) by either actively removing thedissolved gas (degassing) or by increasing the fluid pressure. In oneembodiment, both of these are done concurrently/simultaneously, with theincreased pressure actually increasing the dissolved gas carryingcapacity of the media. The greater the applied pressure, the greater theincrease in media gas carrying capacity, the bigger/faster a bubble canbe crushed. However, there is a practical limit to this.

It has been found that it would be difficult to effectively crushbubbles if the media remained static (did not flow past the bubble). Thereason for this is the relatively long and narrow geometry of themicrochannels. As the media dissolves the bubble, it comes closer andcloser to equilibrium/saturation and cannot dissolve any more gas. Thereis not enough volume of media in the microchannels to fully dissolve thebubbles at “reasonable” applied pressures (not enough gas carryingcapacity). However, by flowing new (fresh), under-saturated media pastthe bubbles, this new media can continue dissolving the bubbles.

Looked at another way, the small geometry of a microchannel puts a limiton the size of the bubble. The bubble is small because the space in themicrochannel is small. Thus, the ability/time to dissolves bubbles isdependent on applied pressure, flow rate, and initial volume of thebubble (the bigger the bubble, the longer it takes to fully dissolve).Using small pressure differentials that generate significant absolutepressure, the bubble comes to equilibration with media very quickly(nearly instantaneously) and completely. In a preferred embodiment, thefollowing conditions are used:

Pressure IN=21 kPa

Pressure OUT=20 kPa

Time bubble CRUSH=2 hrs

These conditions work well in practice (i.e. crushing/dissolving bubbleswithout killing cells). Under these conditions, one should be able tofully remove all the bubbles in 1 hr, but in an abundance of caution,one can run the bubble crush cycle for 2 hrs.

DESCRIPTION OF THE FIGURES

FIG. 1A is an exploded view of one embodiment of the perfusion manifoldassembly showing the cover off of the reservoirs, the reservoirs abovethe backplane, the backplane in fluidic communication with the skirt,the skirt with a side track for engaging a representative microfluidicdevice or “chip” having one or more inlet, outlet and vacuum ports, thechip shown next to (but not in) one embodiment of a chip carrier, thecarrier is configured to support and carrier the chip. FIG. 1B shows thesame embodiment of the perfusion manifold assembly with the cover on andover the reservoirs, and the chip inside the chip carrier fully linkedto the skirt of the perfusion manifold assembly, and thereby in fluidiccommunication with the reservoirs. FIG. 1C shows an exploded view of oneembodiment of the cover assembly comprising a pressure cover or pressurelid, and an associated gasket thereunder.

FIG. 2A shows a side view of one embodiment of a chip carrier (with thechip inside) approaching (but not yet engaging) a side track of a skirtof one embodiment of the perfusion manifold assembly, the carrieraligned at an angle matching an angled front end portion of the sidetrack, the carrier comprising a retention mechanism configured as aupwardly protecting clip. FIG. 2B shows a side view of one embodiment ofa chip carrier (with the chip inside) engaging a side track of a skirtof one embodiment of (but not yet linked to) the perfusion manifoldassembly. FIG. 2C shows a side view of one embodiment of a chip carrier(with the chip inside) fully engaging a side track of a skirt of oneembodiment of (but not yet linked to) the perfusion manifold assembly(with an arrow showing the necessary direction of movement to get a snapfit whereby the retention mechanism will engage to prevent movement).FIG. 2D shows a side view of one embodiment of a chip carrier (with thechip inside) detachably linked to the perfusion manifold assembly, wherethe retention mechanism is engaged to prevent movement. FIG. 2E is asummary slide schematically showing one embodiment of a linking approachto the manifold comprising a sliding action (FIG. 2E-1), pivoting (FIG.2E-2), and snap fit (FIG. 2E-3) in a single action.

FIGS. 3A&B show schematics showing one embodiment of connecting twomicrofluidic devices, resulting in the introduction of air or gasbubbles into the microchannels. FIG. 3A shows two fluidically primeddevices with microchannels that are not yet connected. FIG. 3B shows thedevices of FIG. 3A contacting in a manner that results in theintroduction of air bubbles into the microchannels.

FIG. 4A is a schematic showing the location of a bubble where a chip isengaged by the perfusion manifold assembly (also called the perfusiondisposable or POD). FIG. 4B is a drawing from a photograph of a bubble(see arrow) caught in the perfusion disposable (not the chip) at thelocation circled in FIG. 4A. The perfusion disposable is comprised ofCOP (cyclic polyolefin) with a SEBS (Styrene Ethylene Butylene Styrene)capping layer. COP and SEBS are both substantially less gas permeablethan PDMS.

FIG. 5A is a schematic of an illustrative microfluidic device or“organ-on-chip” device (which can be fabricated out of plastic, such asPDMS) with a mating surface (21). The assembled device in FIG. 5Aincludes a plurality of ports. FIG. 5B shows an exploded view of thedevice of FIG. 5A, showing a tissue-tissue interface simulation region(SR) comprising a membrane, where cell behavior and/or passage of gases,chemicals, molecules, particulates and cells are monitored.

FIG. 6 is a schematic of a “push” based flow approach where fluid flowsfrom a reservoir (on the right) where the fluid is put under highpressure. The fluid exits the reservoir (on the right) and flows in thedirection of the chip (see arrows showing the direction of flow) througha resistor (switchback). There is no pressure applied to the otherreservoir (on the left)

FIG. 7 is a schematic of a “pull” based flow approach where fluid flowsfrom a reservoir (on the left) where the fluid is under no pressure, butwhere the other reservoir (on the right) has low pressure (e.g. becauseof a vacuum). The fluid exits the reservoir (on the left) and flows inthe direction of the chip (see arrows showing the direction of flow).

FIG. 8 is a schematic of a pressure differential (“delta P”) based flowapproach where fluid in both reservoirs are under pressure (P₂ is notzero but is less than P₁). The fluid exits the reservoir (on the right)and flows in the direction of the chip (see arrows showing the directionof flow) through a resistor (switchback).

FIG. 9 show experimental results for reducing bubble volume using thepressure differential based flow approach of FIG. 8. The flow rates inFIG. 9 are given in terms of pressure differentials (delta)—1 kPacorresponds to 40 uL/hr, 1.5 kPa is 60 uL/hr, 3 kPa is 120 uL/hr, 8 kPais 320 uL/hr, 28.3 kPa is 1.13 mL/hr. Pressures applied to the outletsand therefore “felt” by the bubbles are given in terms of “felt”pressure (units of kPa).

FIG. 10 is a chart showing how large, multi-week experiments areperformed with many microfluidic devices or “chips,” underscoring thatthe task of refreshing the media (e.g. every other day or at key timepoints) can be burdensome.

FIGS. 11A&B show the results of an experiment involving the use ofnon-equilibrated media in 19 pods engaging organs-on-chip, with flowrate as the read-out for detecting bubbles. FIG. 11A shows the resultsfor 8 pods and FIG. 11B shows the results for 11 pods (the dotted linesshow the 20% deviation from the bottom average and the top average).

FIGS. 12A&B show plots of pressure versus time in order to test for lidfailure when higher pressures are used for bubble treatment for thingaskets (FIG. 12A) or thick gaskets (FIG. 12B). FIG. 12A shows that, asthe pressure was raised and approached 25-30 kPa, the perfusion systemwith a thin gasket exhibited lid failure. FIG. 12B shows that, as thepressure was raised and approached 33 kPa, the perfusion system withthicker gasket (2-3 times thicker than the thin gasket) did not exhibitlid failure.

FIG. 13 is a bar graph showing axon growth in a microfluidic device overtime (e.g. Day 0, Day 1, Day 4 and Day 5) with 50 μL/hr controlconditions versus 75 μL/hr test conditions.

DEFINITIONS

“Channels” are pathways (whether straight, curved, single, multiple, ina network, etc.) through a medium (e.g., silicon, glass, polymer, etc.)that allow for movement of liquids and gasses. Channels thus can connectother components, i.e., keep components “in communication” and moreparticularly, “in fluidic communication” and still more particularly,“in liquid communication.” Such components include, but are not limitedto, liquid-intake ports and gas vents. Microchannels are channels withdimensions less than 1 millimeter and greater than 1 micron. It is notintended that the present invention be limited to only certainmicrochannel geometries. In one embodiment, a four-sided microchannel iscontemplated. In another embodiment, the microchannel is circular (inthe manner of a tube) with curved walls. In yet another embodiment,combination of circular or straight walls are used.

It is not intended that the present invention be limited by the numberor nature of channels in the microfluidic device. In some embodiments,the surface can be a surface of a fluid-flowing conduit or passagewaydisposed in a solid substrate. In some embodiments, the surface can be asolid surface. For example, in one embodiment, the solid surface can bea wall surface of a fluid channel, e.g., a microfluidic channel.However, the method need not be limited to microchannels, since it willwork in any confined space where fluid flows.

Additionally, the term “microfluidic” as used herein relates tocomponents where moving fluid is constrained in or directed through oneor more channels wherein one or more dimensions are 1 mm or smaller(microscale). Microfluidic channels may be larger than microscale in oneor more directions, though the channel(s) will be on the microscale inat least one direction. In some instances the geometry of a microfluidicchannel may be configured to control the fluid flow rate through thechannel (e.g. increase channel height to reduce shear or resistance).Microfluidic channels can be formed of various geometries to facilitatea wide range of flow rates through the channels.

A “perfusion manifold assembly” is contemplated that allows forperfusion of a microfluidic device, such as an organ on a chipmicrofluidic device comprising cells that mimic cells in an organ in thebody, that is detachably linked with said assembly so that fluid entersports of the microfluidic device from a fluid reservoir, without tubing,at a controllable flow rate. In one embodiment (see FIGS. 1A and 1B),the perfusion manifold assembly comprises i) a cover or lid configuredto serve as the top of ii) one or more fluid reservoirs, iii) a cappinglayer under said fluid reservoir(s), iv) a fluidic backplane under, andin fluidic communication with, said fluid reservoir(s), said fluidicbackplane comprising a resistor, and v) a skirt (for engaging themicrofluidic device). In one embodiment, a combination of pressure andflow reduces bubble volume in a perfusion manifold assembly. In oneembodiment, the perfusion manifold assembly is made of a polymer that isless gas permeable than PDMS.

In one embodiment, the perfusion manifold is linked to a microfluidicdevice (e.g. in fluidic communication therewith). Microfluidic devices(or “chips”) containing living cells recreate the physiologicaltissue-tissue interfaces and permit fluid flow. See U.S. Pat. No.8,647,861, hereby incorporated by reference. Such devices subject thecells to shear stress. In contrast to static 2D culture, microchannelsallow the perfusion of cell culture medium throughout the cell cultureduring in vitro studies and as such offer a more in vivo-like physicalenvironment. In simple terms, an inlet port allows injection of fluidssuch as blood, serum, plasma, cell culture medium (and the like) into amicrofluidic channel or chamber (with or without cells). In oneembodiment, the present invention contemplates a cell-laden microfluidicchannel or chamber. An outlet port then permits the exit of remainingfluid as well as harmful metabolic by-products. In one embodiment, onlyflow is used with media previously under-saturated.

In some embodiments, a bubble is trapped in a microfluidic deviceagainst a polymer that is largely gas impermeable, such as (but notlimited to) a COP. Cyclic olefin copolymers (COCs) and cyclic olefinpolymers (COPs) are very attractive thermoplastic resins with potentialenhanced properties such as outstanding transparency, good heatresistance, low moisture absorption, good chemical resistance, and lowdouble refraction. COCs are obtained through copolymerization ofcycloolefin with ethylene or α-olefin, and commercialized under thetrade names APEL® by Mitsui and TOPAS® by TOPAS advanced polymers (TAP:formerly Ticona and Hoechst). COPs are prepared via ring-openingmetathesis polymerization (ROMP) of cycloolefin followed byhydrogenation, and commercialized under the trade names Zeonex® andZeonor® by Zeon [25] and Arton® by Japan Synthetic Rubber (JSR).

DESCRIPTION OF PREFERRED EMBODIMENTS

Methods of removing gas or air bubbles from a microfluidic device aredescribed, including one or more bubbles in a microchannel of amicrofluidic device. It is not the presence of air, or gas, in themedium which causes the problem. It is the formation of the bubbles fromthese gases which cause the problem. The question is why and how thesebubbles are formed. If the source of bubble formation is established andthen removed, only then this problem can be addressed.

One source of the bubble formation may be explained as follows: cellsare provided nutrients from culture media maintained at 37° C. However,the culture media used are generally stored at room temperature (orless) which is lower than 37° C. When a medium is transferred out ofstorage and heated up to 37° C., there is a change in solubility of thedissolved gasses. The decrease in solubility of the gasses at highertemperatures causes the dissolved gasses to come out of the medium inthe form of tiny bubbles which tend to stick to surfaces of themicrofluidic device housing the cells, including channel surfaces (and,in particular, microdefects in the channel surfaces). While notintending in any way to limit the present invention to any particularmechanism, it is believed that this process of “bubble growth” requiresan initial bubble, sometimes referred to as a nucleation point or “seedbubble,” for the gas in solution to diffuse into and transition fromdissolved gas into non-dissolved gas pockets or bubbles. However, oncethe medium is equilibrated at 37° C. the formation of the bubbles slows.Therefore, one partial answer to the question of why and how the bubblesare formed is because of a transitory stage during the heating processof the culture media.

Up to now, it has been believed that a simple solution to avoid thisproblem is to remove the temperature gradient effect, i.e., avoidtransferring low temperature medium directly into the microfluidicdevice. In other words, one should warm the medium to 37° C. outside themicrofluidic device and/or give sufficient time for the medium toequilibrate in a vessel or reservoir at 37° C. (with moderate stirringif needed). Of course, this takes time and the culture media needs to besterile.

While the practice of de-aeration or “de-gassing” has been introduced toaddress this problem of bubble formation, it is a practice that haspractical limitations. The commonly suggested procedure of de-aerating,which is based on heating/vacuum steps, is oftentimes without ameasurable endpoint and highly dependent on the equipment being used toperform the procedure. Therefore, the de-aeration step will beunpredictable with a high degree of variability stemming from exactprocess parameters and equipment used. Additionally, “de-gassing” canhave the consequence of removing gasses from solution that are needed tomaintain culture, like oxygen (for cellular respiration) and CO₂ (for pHbuffering). Moreover, no matter how reproducible one tries to be withthe de-aeration step, after de-aeration the medium will quickly startequilibrating itself with the atmospheric gasses. Therefore, until thisequilibrium is exactly reached, the system will remain unstable andunreliable.

Where large, multi-week experiments are performed with many microfluidicdevices or “chips,” the task of refreshing the media (e.g. every otherday or at key time points) can be burdensome. This is illustrated inFIG. 10 for a 2 week experiment involving organ-on-chips.

Of course, the physiological environment of the cells in a microfluidicdevice does not require a de-aerated medium. The degassing is only beingdone to address the bubble problem.

This brings one to the question of whether (and to what extent)non-equilibrated and non-degassed culture media can be employed withmicrofluidic devices. In one embodiment, the present inventioncontemplates equilibrating via the process of degassing (physicallyremoving dissolved gas from solution) media before a first pressure/flowcycle—but using non-equilibrated and non-degassed media when replacingmedia thereafter, i.e. during long-term culture. In another embodiment,the present invention contemplates using non-equilibrated andnon-degassed media even in a first pressure/flow cycle (albeit withhigher pressures) whenever culture media is placed into the perfusionmanifold or “pod” reservoir(s). In one embodiment, the present inventioncontemplates adding cold/non-equilibrated media into one or more podreservoirs.

In the first embodiment, culture media is equilibrated and/or de-gassedonce, at the beginning of the experiment, and then a pressure/flowtreatment is utilized for a period of time. Ideally, the period of timeshould be short and insensitive to variability (e.g. 1-2 hours), and thetreatment conditions should allow for operating without unrealisticallyhigh pressures or flow rates. Without intending to limit the inventionin any way to a mechanism of action, it is believed that two forces workin concert to shrink bubbles in such a pressure/flow treatment. First,pressure increases the gas carrying capacity of media. Second, flow(e.g. 40 μL/hr) provides fresh (undersaturated) media into which thebubbles dissolve. It has been empirically observed that oversaturatedmedia cannot grow bubbles that do not exist in the first place.Thereafter, culture media would not need to be equilibrated or degassedwhen replenishing media. Said another way, the single pressure/flowtreatment removes the bubbles (or nucleation points/seed bubbles) andthe use of oversaturated media thereafter will not bring them back. Inthis embodiment, non-equilibrated media can be used when refilling inletreservoirs AFTER a single pressure/flow cycle has successfullyeliminated system bubbles. The benefit of this approach is that itsolves the bubble problem, while decreasing the number of times culturemedia must be equilibrated and/or degassed.

In the second embodiment, culture media is not equilibrated (i.e. it isnon-equilibrated culture media) and has not gone the physical removal ofdissolved gas via degassing. In order for this to work, it has beenmathematically determined via physical principals and confirmedexperimentally that one can increase the pressure (e.g. by 13 kPa ormore) during the pressure/flow cycle (e.g. increase from 20 kPa to 33kPa or more). While not intending to be limited to any particularmechanism, it is believed that this increased pressure increasesnon-equilibrated media gas carrying capacity to match equilibrated mediagas carrying capacity, making the pressure/flow cycle as effective(theoretically) as with non-equilibrated media. The increased pressurecan put a strain on the microfluidic system. However, it has beenempirically determined that a thicker gasket for the perfusion manifoldis one solution to avoiding leaks associated with the increasedpressure. Optionally, increased flow rates (from 50 to 75 μL/hr) canalso be used (and provide some benefit in terms of robustness ofeliminating bubbles) since it has been empirically found that the cellscan tolerate the increased flow. With regard to increased pressure, itappears that the pressure differential between the reservoirs (i.e. theinlet and outlet reservoirs) is more important to the viability of thecells than the actual pressures employed. It has been empirically foundthat pressure differentials of 2 kPa or less are useful, more preferably1.5 kPa or less, still more preferably 1.0 kPa or less.

Description of Exemplary Microfluidic Devices

In one embodiment (as shown in FIG. 1A), the perfusion manifold assemblyor POD (10) comprises i) a cover or lid (11) configured to serve as totop of ii) one or more fluid reservoirs (12), iii) a capping layer (13)under said fluid reservoir(s), iv) a fluidic backplane (14) under, andin fluidic communication with, said fluid reservoir(s), said fluidicbackplane comprising a fluidic resistor, and v) a skirt (15) forengaging the microfluidic device (16) which is preferably positioned ina carrier (17). In one embodiment, the carrier (17) has a tab or othergripping platform (18), a retention mechanism such as a clip (19), and avisualization cutout (20) for imaging the chip. In one embodiment, thefluidic resistor comprises a series of switchbacks or serpentine fluidchannel (not shown).

FIG. 1C shows an exploded view of one embodiment of the cover assembly(11) comprising a pressure cover or pressure lid. In the illustratedembodiment, the pressure lid comprises a plurality of ports (e.g.through-hole ports) associated with filters (38) and corresponding holes(39) in a gasket (37). The illustrated design of the holes in the gasketis intended to permit the gasket to aid in retaining the illustratedfilters in position. In alternative embodiments, gasket openings mayemploy a shape different from openings in the lid. For example, thegasket can be shaped to follow the contour of one or more reservoirswith which it is intended to form a fluidic or pressure seal. In someembodiments, a plurality of gaskets may be employed. In a preferredembodiment, a thicker gasket may be employed (in order to avoid leakingunder the higher pressures described herein to treat bubbles). In someembodiments, the filters and/or gasket may be fixed using an adhesive,heat stacking, bonding (ultrasonic, solvent-assisted, laser welding),clamped, or captured by elements of the lid and/or an additionalsubstrate. Although the illustrated pressure lid comprises through-holeports, alternative embodiments comprise one or more channels that routeat least one top-surface port to one or more bottom surface ports, whichneed not be directly underneath the top-surface port.

In one embodiment, the microfluidic device is detachably linked with themanifold assembly by a clipping mechanism that temporarily “locks” themicrofluidic device, including organ-on-chip devices, in place (FIGS.2A, 2B, 2C, 2D and 2E). In one embodiment, the clipping or “snapfitting” involves a projection on the carrier (19) which serves as aretention mechanism when the microfluidic device is positioned. In oneembodiment, the clipping mechanism is similar to the interlockingplastic design of a Lego™ chip and comprises a straight-down clip.However, in another embodiment, the clipping mechanism is triggered onlyafter the microfluidic device, or more preferably, the carriercomprising the microfluidic device, engages the perfusion manifoldassembly (or cartridge) on a guide rail, side slot, internal or externaltrack (25) or other mechanism that provides a stable glide path for thedevice as it is conveyed (e.g. by machine or by hand) into position. Theguide rail, side slot, internal or external track (25) or othermechanism can be, but need not be, strictly linear and can be positionedin a projecting member or skirt attached to the main body of themanifold assembly. In one embodiment, the beginning portion of the guiderail, side slot, internal or external track or other mechanism comprisesan angled slide (27) which provides a larger opening for easier initialpositioning, followed by a linear or essentially linear portion (28). Inone embodiment, the end portion (29) (close to the corresponding portsof the assembly) of an otherwise linear (or essentially linear) guiderail, side slot, internal track or other mechanism is angled (or curves)upward so that there is a combination of linear movement (e.g.initially) and upward movement to achieve linking.

The POD has a few features that help reduce bubble introduction: 1) theclip has a very smooth engagement—rough engagements and/or jerkingmotions can introduce bubbles, and 2) the POD diameter going to the chiphas been minimized to reduce bubble trapping upon initial filling of thePOD—this minimizes dead volume where pockets of air can get trapped.

The advantage of the carrier is that the surfaces of the microfluidicdevice need not be touched during the detachable linage with themanifold assembly. The carrier can have a plate, platform, handle orother mechanism for gripping the carrier (18), without contacting themating surface (21) of the microfluidic device (16). The retentionmechanism (19) can comprise a projection, hook, latch or lip thatengages one or more portions of the manifold assembly, and morepreferably the skirt of the manifold assembly, to provide a “snap fit.”

FIGS. 5A&B show schematics of illustrative microfluidic devices or“organ-on-chip” devices. The assembled device in FIG. 5A includes aplurality of ports. FIG. 5B shows an exploded view of the device of FIG.5A, showing a tissue-tissue interface simulation region (“SR”)comprising a membrane (100), where cell behavior and/or passage ofgases, chemicals, molecules, particulates and cells are monitored.

Bubbles can be introduced when a chip is engaged by the perfusionmanifold assembly (also called the perfusion disposable). FIGS. 4A and4B underscore this point, showing a bubble (see arrow) caught in theperfusion disposable (not the chip) at the location circled in FIG. 4A.Any one of the embodiments of the methods described above for combiningpressure and flow may be used to reduce the volume of such bubbles insuch microfluidic devices.

In one embodiment, the POD is positioned on the culture module and thepressure surface of the culture module move down to engage the cover orlid (11) of the perfusion manifold assembly (10). Embodiments of aculture module are described in U.S. patent application Ser. No.15/248,509, hereby incorporated by reference. As shown in FIG. 1C, thecover or lid comprises ports such as through-hole ports (36) that areengaged by corresponding pressure points on the pressure surface of theculture module. These ports (36), when engaged, transmit appliedpressure inward through the cover and through a gasket (37) and applythe pressure to the fluid in the reservoirs (12) of the perfusionmanifold assembly (10). Thus, in this embodiment, pressure is appliedthrough the lid (11) and the lid seals against the reservoir(s). Forexample, when on applies 1 kPa, this nominal pressure results, in oneembodiment, in a flow rate of approximately 30-40 uL/hr.

EXPERIMENTAL Example 1

In this experiment, 19 pods engaging organs-on-chip (in this case,microfluidic devices with viable intestinal cells growing on a membranein a microchannel) were utilized. They were previously running for 6days, with no history of bubbles. In the test groups, inlet reservoirswere filled with cold media (4° C.) on days 0 and 2, warm media (notequilibrated) on day 7. Flow was measured daily as a read-out (sincebubbles disrupt flow and thus a change in flow would indicate bubbles);in addition, the pods/chips were visually inspected for bubbles. Theresults are shown in FIGS. 11A and 11B. No bubble growth/generation wasobserved or detected in any pod/chip when using non-equilibrated media(warm or cold) over 9 days.

Example 2

In this experiment, one embodiment of the perfusion system's ability towithstand higher pressures was tested (in order to see if working withnon-equilibrated media at higher pressures is feasible). Variouscomponents on the POD (FIGS. 1A and 1B) were examined, including the lid(FIG. 1C) and other interfaces (e.g. gaskets, bonded components, etc.).In addition, components of a culture module (described in U.S. patentapplication Ser. No. 15/248,509, hereby incorporated by reference) wereexamined in these pressure tests (e.g. the manifold, valves andjunctions). As the pressure was raised and approached 25-30 kPa, theperfusion system with a thin gasket (FIG. 1C, element 37) exhibited lidfailure and leakage (FIG. 12A). However, when a thicker gasket (2-3times thicker than the thin gasket), there was no lid failure or leakageeven at 33 kPa (FIG. 12B). With the thicker gasket, the perfusion systemcan withstand ˜34 kPa on the inlet and ˜33 kPa on the outlet.

Example 3

In this experiment, higher flow rates were tested to determine whetherthere are negative cell effects. More specifically, the viability andfunction of human primary human motor neurons maintained after 7 dayswas assessed (since they are relatively sensitive to culture conditionsand shear forces). Flow rates of 50 (control) to 75 μL/hr (test) wereused to perfuse the cells in a microfluidic chip engaged in a POD (FIGS.1A and 1B), with the POD engaged with a culture module (described inU.S. patent application Ser. No. 15/248,509, hereby incorporated byreference). The outlet pressure was 20.0 kPa+/−0.5 kPa. The inletpressure minus the outlet pressure [(Inlet Pressure)−(Outlet Pressure)]was 1.5 kPa+/−0.5 kPa. Primary motor neurons were seeded (on day zero)and cultured for 7 days, and the medium was replenished every two days.A pressure/flow process was run on Days 1 and 5 to treat bubbles. Coldmedia was placed in the POD reservoir on Days 3 and 5. Cells were imaged(phase contrast captured at 200×).

Axon growth was observed in both control and experimental conditions.FIG. 13 is a bar graph showing the results at Day 0, Day 1, Day 4 andDay 5. Results are average ±SE in 2 independent PODs for the 50 μLcondition and in 3 independent PODs for the 75 μL condition.

Motor neurons were stained (after 7 days) with Hoechst 33342 (blue),which indicates cell nuclei and Tuj-1 (green), which marks β-Tubulin 3—aprotein vital to microtubule stability and transport in the axon ofneurons. Neuron staining revealed well-developed neuronal networks inthe control (50 μL/hr) and in the test (75 μL/hr) (data not shown). Insum, the experiment showed that 1) motor neurons are capable of handlingelevated flow rates, i.e. flow rates that help to facilitate bubbleremoval 2) capable of handling multiple pressure/flow cycles at 20 kPaapplied pressure and that 3) the use of cold media to refill inletreservoirs during normal media refresh/addition steps did not cause theformation of bubbles after the initial pressure/flow step to removesystem bubbles.

1. A method for establishing a fluidic connection, comprising: a)providing a first substrate comprising a first fluidic port, a secondsubstrate comprising a second fluidic port; b) aligning the first andsecond sets of fluidic ports; c) contacting the first and second fluidicports to establish a fluidic connection under conditions such that abubble forms, said bubble having a volume; and d) flowing fluid underpressure through said first or second port under conditions such thatsaid bubble volume is reduced.
 2. The method of claim 1, wherein saidfirst substrate comprises a guide mechanism adapted to guide the secondsubstrate.
 3. The method of claim 2, further comprising prior to step b)engaging the second substrate with the guide mechanism.
 4. The method ofclaim 2, wherein said aligning of step b) is performed with the guidemechanism.
 5. The method of claim 4, wherein said guide mechanismcomprises a guide track positioned on said first substrate, said guidetrack configured to engage a portion of said second substrate.
 6. Themethod of claim 1, wherein said bubble of step c) is positioned againsta polymer that is substantially gas impermeable.
 7. The method of claim1, wherein said bubble is a gas bubble.
 8. The method of claim 7,wherein said gas is oxygen, nitrogen or a mixture thereof.
 9. The methodof claim 1, wherein said bubble is an air bubble.
 10. The method ofclaim 1, wherein said flowing of fluid is at a flow rate of 40 uL/hr.11. The method of claim 1, wherein said first substrate comprises achannel in fluidic communication with said port.
 12. The method of claim11, wherein said channel is a microchannel.
 13. The method of claim 11,wherein said first substrate is a perfusion manifold.
 14. The method ofclaim 13, wherein said second substrate is a microfluidic device. 15.The method of claim 14, wherein said perfusion manifold engages saidmicrofluidic device at step c).
 16. The method of claim 14, wherein saidmicrofluidic device comprises a microchannel, said microchannelcomprising living cells, and said fluid comprises media supplied to saidcells.
 17. The method of claim 16, wherein said media prior to step d)was degassed.
 18. The method of claim 16, wherein said media of step d)is unsaturated.
 19. The method of claim 16, wherein said media prior tostep d) was not degassed.
 20. The method of claim 1, wherein step d) isperformed for at least one 1 hour.
 21. The method of claim 20, whereinstep d) is performed for 2 hours.
 22. The method of claim 20, furthercomprising e) introducing fluid into said microchannel, wherein saidfluid has not been degassed.